The long-term objective of this proposal is to determine the mechanism by which the packaging of a gene in heterochromatin leads to stable inactivation. This type of "off" regulation is clearly of functional important in chromosome imprinting, and may extend to the regulation of the homeotic loci, whose on/off pattern is critical for normal develop- ment in all higher organisms. Drosophila will be used throughout this study because of the ease of manipulation and monitoring of Position effect variegation, one type of epigenetic down-regulation that appears to reflect the packaging of the test gene. We have identified a heterochromatin-associated protein, HP1, which plays a role in this gene inactivation; mutations in HP1 result in suppression of position effect variegation in Drosophila. Our first aim is to identify proteins which interact with HP1, using biochemical and genetic approaches. Interacting proteins will be sought using (a) a genetic screen in Drosophila; (b) coprecipitation with anti-HP1 antibodies; and (c) a screen in yeast that requires protein-protein interactions to score a positive result (Fields and Song, 1989, Nature 340, 245-246). In the genetic screen, missense mutations, including temperature-sensitive alleles of HP1, are being sought, as well as noncomplementing second-site mutations. Interacting proteins identified will be characterized biochemically and genetically to determine their role in heterochromatin formation. In parallel with the study of HP1, we are carrying out studies of the nucleosomal and higher order packaging of transgenes inserted into heterochromatin using P-element mediated transformation. Two different genes are being used, hsp26, a heat shock gene whose 5' upstream regulatory region is normally preset in an accessible chromatin structure prior to gene activation, and a CAT reporter gene under the control of glucocorticoid response elements, a gene likely to be characteristic of those whose 5' upstream regulatory region is packaged in a nucleosomal array and must be remodeled for activation. We will characterize the chromatin structure of these genes when they are packaged in heterochromatin, in comparison to their packaging in euchromatin, in order to learn more about the inactivation process. Having such specific test genes in a heterochromatin environment will also be invaluable in assessing the role of the chromosomal proteins identified above. These studies should move us much closer to an understanding of the nature and regulatory role of higher order chromatin structure.